Modified nucleosides or nucleotides

ABSTRACT

Some embodiments described herein relate to modified nucleotide and nucleoside molecules with novel 3′-hydroxy protecting groups. Also provided herein are methods to prepare such modified nucleotide and nucleoside molecules and sequencing by synthesis processes using such modified nucleotide and nucleoside molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/444,826, filed Feb. 28, 2017 and to be issued as U.S. Pat. No.10,407,721, which is a continuation of U.S. application Ser. No.14/771,481, filed Aug. 28, 2015, now U.S. Pat. No. 9,593,373 issued onMar. 14, 2017, which is the U.S. National Phase under 35 U.S.C. § 371 ofInternational Application No. PCT/EP2013/055466, entitled “MODIFIEDNUCLEOSIDES OR NUCLEOTIDES,” filed Mar. 15, 2013, and published inEnglish on Sep. 18, 2014 as WO 2014/139596 A1, each of which is herebyincorporated by reference in its entirety.

BACKGROUND Field of the Invention

Some embodiments described herein relate to modified nucleotides ornucleosides comprising 3′-hydroxy protecting groups and their use inpolynucleotide sequencing methods. Some embodiments described hereinrelate to method of preparing the 3′-hydroxy protected nucleotides ornucleosides.

Description of the Related Art

Advances in the study of molecules have been led, in part, byimprovement in technologies used to characterize the molecules or theirbiological reactions. In particular, the study of the nucleic acids DNAand RNA has benefited from developing technologies used for sequenceanalysis and the study of hybridization events.

An example of the technologies that have improved the study of nucleicacids is the development of fabricated arrays of immobilized nucleicacids. These arrays consist typically of a high-density matrix ofpolynucleotides immobilized onto a solid support material. See, e.g.,Fodor et al., Trends Biotech. 12: 19-26, 1994, which describes ways ofassembling the nucleic acids using a chemically sensitized glass surfaceprotected by a mask, but exposed at defined areas to allow attachment ofsuitably modified nucleotide phosphoramidites. Fabricated arrays canalso be manufactured by the technique of “spotting” knownpolynucleotides onto a solid support at predetermined positions (e.g.,Stimpson et al., Proc. Natl. Acad. Sci. 92: 6379-6383, 1995).

One way of determining the nucleotide sequence of a nucleic acid boundto an array is called “sequencing by synthesis” or “SBS”. This techniquefor determining the sequence of DNA ideally requires the controlled(i.e., one at a time) incorporation of the correct complementarynucleotide opposite the nucleic acid being sequenced. This allows foraccurate sequencing by adding nucleotides in multiple cycles as eachnucleotide residue is sequenced one at a time, thus preventing anuncontrolled series of incorporations occurring. The incorporatednucleotide is read using an appropriate label attached thereto beforeremoval of the label moiety and the subsequent next round of sequencing.

In order to ensure only a single incorporation occurs, a structuralmodification (“protecting group”) is added to each labeled nucleotidethat is added to the growing chain to ensure that only one nucleotide isincorporated. After the nucleotide with the protecting group has beenadded, the protecting group is then removed, under reaction conditionswhich do not interfere with the integrity of the DNA being sequenced.The sequencing cycle can then continue with the incorporation of thenext protected, labeled nucleotide.

To be useful in DNA sequencing, nucleotides, and more usually nucleotidetriphosphates, generally require a 3′-hydroxy protecting group so as toprevent the polymerase used to incorporate it into a polynucleotidechain from continuing to replicate once the base on the nucleotide isadded. There are many limitations on types of groups that can be addedonto a nucleotide and still be suitable. The protecting group shouldprevent additional nucleotide molecules from being added to thepolynucleotide chain whilst simultaneously being easily removable fromthe sugar moiety without causing damage to the polynucleotide chain.Furthermore, the modified nucleotide needs to be tolerated by thepolymerase or other appropriate enzyme used to incorporate it into thepolynucleotide chain. The ideal protecting group therefore exhibits longterm stability, be efficiently incorporated by the polymerase enzyme,cause blocking of secondary or further nucleotide incorporation and havethe ability to be removed under mild conditions that do not cause damageto the polynucleotide structure, preferably under aqueous conditions.

Reversible protecting groups have been described previously. Forexample, Metzker et al., (Nucleic Acids Research, 22 (20): 4259-4267,1994) discloses the synthesis and use of eight 3′-modified2-deoxyribonucleoside 5′-triphosphates (3′-modified dNTPs) and testingin two DNA template assays for incorporation activity. WO 2002/029003describes a sequencing method which may include the use of an allylprotecting group to cap the 3′-OH group on a growing strand of DNA in apolymerase reaction.

In addition, we previously reported the development of a number ofreversible protecting groups and methods of deprotecting them under DNAcompatible conditions in International Application Publication No. WO2004/018497, which is hereby incorporated by reference in its entirety.

SUMMARY

Some embodiments described herein relate to a modified nucleotide ornucleoside molecule comprising a purine or pyrimidine base and a riboseor deoxyribose sugar moiety having a removable 3′-hydroxy protectinggroup forming a structure —O—C(R)₂N₃ covalently attached to the3′-carbon atom, wherein

-   -   R is selected from the group consisting of hydrogen,        —C(R¹)_(m)(R²)_(n), —C(═O)OR³, —C(═O)NR⁴R⁵,        —C(R⁶)₂O(CH₂)_(p)NR⁷R⁸ and —C(R⁹)₂O-Ph-C(═O)NR¹⁰R¹¹;    -   each R¹ and R² is independently selected from hydrogen,        optionally substituted alkyl or halogen;    -   R³ is selected from hydrogen or optionally substituted alkyl;    -   each R⁴ and R⁵ is independently selected from hydrogen,        optionally substituted alkyl, optionally substituted aryl,        optionally substituted heteroaryl, or optionally substituted        aralkyl;    -   each R⁶ and R⁹ is selected from hydrogen, optionally substituted        alkyl or halogen;    -   each R⁷, R⁸, R¹⁰ and R¹¹ is independently selected from        hydrogen, optionally substituted alkyl, optionally substituted        aryl, optionally substituted heteroaryl, or optionally        substituted aralkyl;    -   m is an integer of 0 to 3; and    -   n is an integer of 0 to 3; provided that the total of m+n equals        to 3; and    -   p is an integer of 0 to 6; provided that    -   R¹ and R² cannot both be halogen; and    -   at least one R is not hydrogen.

Some embodiments described herein relate to a method of preparing agrowing polynucleotide complementary to a target single-strandedpolynucleotide in a sequencing reaction, comprising incorporating amodified nucleotide molecule described herein into the growingcomplementary polynucleotide, wherein the incorporation of the modifiednucleotide prevents the introduction of any subsequent nucleotide intothe growing complementary polynucleotide.

Some embodiments described herein relate to a method for determining thesequence of a target single-stranded polynucleotide, comprisingmonitoring the sequential incorporation of complementary nucleotides,wherein at least one complementary nucleotide incorporated is a modifiednucleotide molecule described herein; and detecting the identity of themodified nucleotide molecule. In some embodiments, the incorporation ofthe modified nucleotide molecule is accomplished by a terminaltransferase, a terminal polymerase or a reverse transcriptase.

Some embodiments described herein relate to a kit comprising a pluralityof modified nucleotide or nucleoside molecule described herein, andpackaging materials therefor. In some embodiments, the identity of themodified nucleotide is determined by detecting the detectable labellinked to the base. In some such embodiments, the 3′-hydroxy protectinggroup and the detectable label are removed prior to introducing the nextcomplementary nucleotide. In some such embodiments, the 3′-hydroxyprotecting group and the detectable label are removed in a single stepof chemical reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a variety of 3′-OH protecting groups.

FIG. 1B illustrated the thermal stability of various 3′-OH protectinggroups.

FIG. 2A illustrates the deprotection rate curve of three different 3′-OHprotecting groups.

FIG. 2B shows a chart of the deprotection half time of three different3′-OH protecting groups.

FIG. 3 shows the phasing and prephasing values of various modifiednucleotide with a thermally stable 3′-OH protecting group in comparisonand the standard protecting group.

FIG. 4A shows the 2×400 bp sequencing data of mono-F ffNs-A-isomer inincorporation mix (IMX).

FIG. 4B shows the 2×400 bp sequencing data of mono-F ffNs-B-isomer inincorporation mix (IMX).

DETAILED DESCRIPTION

One embodiment is a modified nucleotide or nucleoside comprising a 3′-OHprotecting group. In one embodiment, the 3′-OH protecting group is amonofluoromethyl substituted azidomethyl protecting group. In anotherembodiment, the 3′-OH protecting group is a C-amido substitutedazidomethyl protecting group. Still another embodiment relates tomodified nucleotides having difluoromethyl substituted azidomethyl 3′-OHprotecting groups.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art. The use of the term “including” as well as other forms, suchas “include”, “includes,” and “included,” is not limiting. The use ofthe term “having” as well as other forms, such as “have”, “has,” and“had,” is not limiting. As used in this specification, whether in atransitional phrase or in the body of the claim, the terms “comprise(s)”and “comprising” are to be interpreted as having an open-ended meaning.That is, the above terms are to be interpreted synonymously with thephrases “having at least” or “including at least.” For example, whenused in the context of a process, the term “comprising” means that theprocess includes at least the recited steps, but may include additionalsteps. When used in the context of a compound, composition, or device,the term “comprising” means that the compound, composition, or deviceincludes at least the recited features or components, but may alsoinclude additional features or components.

As used herein, common organic abbreviations are defined as follows:

-   -   Ac Acetyl    -   Ac₂O Acetic anhydride    -   aq. Aqueous    -   Bn Benzyl    -   Bz Benzoyl    -   BOC or Boc tert-Butoxycarbonyl    -   Bu n-Butyl    -   cat. Catalytic    -   Cbz Carbobenzyloxy    -   ° C. Temperature in degrees Centigrade    -   dATP Deoxyadenosine triphosphate    -   dCTP Deoxycytidine triphosphate    -   dGTP Deoxyguanosine triphosphate    -   dTTP Deoxythymidine triphosphate    -   ddNTP(s) Dideoxynucleotide(s)    -   DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene    -   DCA Dichloroacetic acid    -   DCE 1,2-Dichloroethane    -   DCM Methylene chloride    -   DIEA Diisopropylethylamine    -   DMA Dimethylacetamide    -   DME Dimethoxyethane    -   DMF N,N′-Dimethylformamide    -   DMSO Dimethylsulfoxide    -   DPPA Diphenylphosphoryl azide    -   Et Ethyl    -   EtOAc Ethyl acetate    -   ffN Fully functional nucleotide    -   g Gram(s)    -   GPC Gel permeation chromatography    -   h or hr Hour(s)    -   iPr Isopropyl    -   KPi 10 mM potassium phosphate buffer at pH 7.0    -   KPS Potassium persulfate    -   IPA Isopropyl Alcohol    -   IMX Incorporation mix    -   LCMS Liquid chromatography-mass spectrometry    -   LDA Lithium diisopropylamide    -   m or min Minute(s)    -   mCPBA meta-Chloroperoxybenzoic Acid    -   MeOH Methanol    -   MeCN Acetonitrile    -   Mono-F —CH₂F    -   Mono-F ffN modified nucleotides with —CH₂F substituted on        methylene position of azidomethyl 3′-OH protecting group    -   mL Milliliter(s)    -   MTBE Methyl tertiary-butyl ether    -   NaN₃ Sodium Azide    -   NHS N-hydroxysuccinimide    -   PG Protecting group    -   Ph Phenyl    -   ppt Precipitate    -   rt Room temperature    -   SBS Sequencing by Synthesis    -   TEA Triethylamine    -   TEMPO (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl    -   TCDI 1,1′-Thiocarbonyl diimidazole    -   Tert, t tertiary    -   TFA Trifluoracetic acid    -   THF Tetrahydrofuran    -   TEMED Tetramethylethylenediamine    -   μL Microliter(s)

As used herein, the term “array” refers to a population of differentprobe molecules that are attached to one or more substrates such thatthe different probe molecules can be differentiated from each otheraccording to relative location. An array can include different probemolecules that are each located at a different addressable location on asubstrate. Alternatively or additionally, an array can include separatesubstrates each bearing a different probe molecule, wherein thedifferent probe molecules can be identified according to the locationsof the substrates on a surface to which the substrates are attached oraccording to the locations of the substrates in a liquid. Exemplaryarrays in which separate substrates are located on a surface include,without limitation, those including beads in wells as described, forexample, in U.S. Pat. No. 6,355,431 B1, US 2002/0102578 and PCTPublication No. WO 00/63437. Exemplary formats that can be used in theinvention to distinguish beads in a liquid array, for example, using amicrofluidic device, such as a fluorescent activated cell sorter (FACS),are described, for example, in U.S. Pat. No. 6,524,793. Further examplesof arrays that can be used in the invention include, without limitation,those described in U.S. Pat. Nos. 5,429,807; 5,436,327; 5,561,071;5,583,211; 5,658,734; 5,837,858; 5,874,219; 5,919,523; 6,136,269;6,287,768; 6,287,776; 6,288,220; 6,297,006; 6,291,193; 6,346,413;6,416,949; 6,482,591; 6,514,751 and 6,610,482; and WO 93/17126; WO95/11995; WO 95/35505; EP 742 287; and EP 799 897.

As used herein, the term “covalently attached” or “covalently bonded”refers to the forming of a chemical bonding that is characterized by thesharing of pairs of electrons between atoms. For example, a covalentlyattached polymer coating refers to a polymer coating that forms chemicalbonds with a functionalized surface of a substrate, as compared toattachment to the surface via other means, for example, adhesion orelectrostatic interaction. It will be appreciated that polymers that areattached covalently to a surface can also be bonded via means inaddition to covalent attachment.

As used herein, any “R” group(s) such as, without limitation, R², R³,R⁴, R⁵, R⁶, R⁷, and R⁸ represent substituents that can be attached tothe indicated atom. An R group may be substituted or unsubstituted. Iftwo “R” groups are described as being “taken together” the R groups andthe atoms they are attached to can form a cycloalkyl, aryl, heteroaryl,or heterocycle. For example, without limitation, if R² and R³, or R²,R³, or R⁴, and the atom to which it is attached, are indicated to be“taken together” or “joined together” it means that they are covalentlybonded to one another to form a ring, an example of which is set forthbelow:

Whenever a group is described as being “optionally substituted” thatgroup may be unsubstituted or substituted with one or more of theindicated substituents. Likewise, when a group is described as being“unsubstituted or substituted” if substituted, the substituent may beselected from one or more the indicated substituents. If no substituentsare indicated, it is meant that the indicated “optionally substituted”or “substituted” group may be individually and independently substitutedwith one or more group(s) individually and independently selected from agroup of functionalies including, but not limited to, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl,hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio,arylthio, cyano, halogen, thiocarbonyl, O-carbamyl, N-carbamyl,O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido,N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato,thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl,haloalkyl, haloalkoxy, trihalomethanesulfonyl,trihalomethanesulfonamido, amino, mono-substituted amino group,di-substituted amino group, and protected derivatives thereof.

As used herein, “alkyl” refers to a straight or branched hydrocarbonchain that comprises a fully saturated (no double or triple bonds)hydrocarbon group. In some embodiments, the alkyl group may have 1 to 20carbon atoms (whenever it appears herein, a numerical range such as “1to 20” refers to each integer in the given range inclusive of theendpopints; e.g., “1 to 20 carbon atoms” means that the alkyl group mayconsist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up toand including 20 carbon atoms, although the present definition alsocovers the occurrence of the term “alkyl” where no numerical range isdesignated). The alkyl group may also be a medium size alkyl havingabout 7 to about 10 carbon atoms. The alkyl group can also be a loweralkyl having 1 to 6 carbon atoms. The alkyl group of the compounds maybe designated as “C₁-C₄ alkyl” or similar designations. By way ofexample only, “C₁-C₄ alkyl” indicates that there are one to four carbonatoms in the alkyl chain, i.e., the alkyl chain is selected from methyl,ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.Typical alkyl groups include, but are in no way limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, andhexyls. The alkyl group may be substituted or unsubstituted.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Analkenyl group may be unsubstituted or substituted.

As used herein, “alkynyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more triple bonds. Analkynyl group may be unsubstituted or substituted.

As used herein, “cycloalkyl” refers to a completely saturated (no doubleor triple bonds) mono- or multi-cyclic hydrocarbon ring system. Whencomposed of two or more rings, the rings may be joined together in afused fashion. Cycloalkyl groups can contain 3 to 10 atoms in thering(s). In some embodiments, cycloalkyl groups can contain 3 to 8 atomsin the ring(s). A cycloalkyl group may be unsubstituted or substituted.Typical cycloalkyl groups include, but are in no way limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, andcyclooctyl.

As used herein, “aryl” refers to a carbocyclic (all carbon) monocyclicor multicyclic aromatic ring system (including, e.g., fused, bridged, orspiro ring systems where two carbocyclic rings share a chemical bond,e.g., one or more aryl rings with one or more aryl or non-aryl rings)that has a fully delocalized pi-electron system throughout at least oneof the rings. The number of carbon atoms in an aryl group can vary. Forexample, in some embodiments, the aryl group can be a C₆-C₁₄ aryl group,a C₆-C₁₀ aryl group, or a C₆ aryl group. Examples of aryl groupsinclude, but are not limited to, benzene, naphthalene, and azulene. Anaryl group may be substituted or unsubstituted.

As used herein, “heterocyclyl” refers to ring systems including at leastone heteroatom (e.g., O, N, S). Such systems can be unsaturated, caninclude some unsaturation, or can contain some aromatic portion, or beall aromatic. A heterocyclyl group may be unsubstituted or substituted.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system (a ring system having a least one ring with a fullydelocalized pi-electron system) that contain(s) one or more heteroatoms,that is, an element other than carbon, including but not limited to,nitrogen, oxygen, and sulfur, and at least one aromatic ring. The numberof atoms in the ring(s) of a heteroaryl group can vary. For example, insome embodiments, a heteroaryl group can contain 4 to 14 atoms in thering(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s).Furthermore, the term “heteroaryl” includes fused ring systems where tworings, such as at least one aryl ring and at least one heteroaryl ring,or at least two heteroaryl rings, share at least one chemical bond.Examples of heteroaryl rings include, but are not limited to, furan,furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole,benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole,1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole,benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole,benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole,tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine,pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline,and triazine. A heteroaryl group may be substituted or unsubstituted.

As used herein, “heteroalicyclic” or “heteroalicyclyl” refers to three-,four-, five-, six-, seven-, eight-, nine-, ten-, up to 18-memberedmonocyclic, bicyclic, and tricyclic ring system wherein carbon atomstogether with from 1 to 5 heteroatoms constitute said ring system. Aheterocycle may optionally contain one or more unsaturated bondssituated in such a way, however, that a fully delocalized pi-electronsystem does not occur throughout all the rings. The heteroatoms areindependently selected from oxygen, sulfur, and nitrogen. A heterocyclemay further contain one or more carbonyl or thiocarbonylfunctionalities, so as to make the definition include oxo-systems andthio-systems such as lactams, lactones, cyclic imides, cyclicthioimides, and cyclic carbamates. When composed of two or more rings,the rings may be joined together in a fused fashion. Additionally, anynitrogens in a heteroalicyclic may be quaternized. Heteroalicyclyl orheteroalicyclic groups may be unsubstituted or substituted. Examples ofsuch “heteroalicyclic” or “heteroalicyclyl” groups include but are notlimited to, 1,3-dioxin, 1,3-dioxane, 1,4-dioxane, 1,2-dioxolane,1,3-dioxolane, 1,4-dioxolane, 1,3-oxathiane, 1,4-oxathiin,1,3-oxathiolane, 1,3-dithiole, 1,3-dithiolane, 1,4-oxathiane,tetrahydro-1,4-thiazine, 2H-1,2-oxazine, maleimide, succinimide,barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin,dihydrouracil, trioxane, hexahydro-1,3,5-triazine, imidazoline,imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine,oxazolidinone, thiazoline, thiazolidine, morpholine, oxirane, piperidineN-Oxide, piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione,4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine,tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine,thiamorpholine sulfoxide, thiamorpholine sulfone, and their benzo-fusedanalogs (e.g., benzimidazolidinone, tetrahydroquinoline,3,4-methylenedioxyphenyl).

As used herein, “aralkyl” and “aryl(alkyl)” refer to an aryl groupconnected, as a substituent, via a lower alkylene group. The loweralkylene and aryl group of an aralkyl may be substituted orunsubstituted. Examples include but are not limited to benzyl,2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

As used herein, “heteroaralkyl” and “heteroaryl(alkyl)” refer to aheteroaryl group connected, as a substituent, via a lower alkylenegroup. The lower alkylene and heteroaryl group of heteroaralkyl may besubstituted or unsubstituted. Examples include but are not limited to2-thienylalkyl, 3-thienylalkyl, furylalkyl, thienylalkyl, pyrrolylalkyl,pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl, and theirbenzo-fused analogs.

As used herein, “alkoxy” refers to the formula —OR wherein R is analkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or acycloalkynyl is defined as above. A non-limiting list of alkoxys ismethoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,iso-butoxy, sec-butoxy, and tert-butoxy. An alkoxy may be substituted orunsubstituted.

As used herein, a “C-amido” group refers to a “—C(═O)N(R_(a)R_(b))”group in which R_(a) and R_(b) can be independently hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. AC-amido may be substituted or unsubstituted.

As used herein, an “N-amido” group refers to a “RC(═O)N(R_(a))—” groupin which R and R_(a) can be independently hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. An N-amido may besubstituted or unsubstituted.

The term “halogen atom”, “halogen” or “halo” as used herein, means anyone of the radio-stable atoms of column 7 of the Periodic Table of theElements, such as, fluorine, chlorine, bromine, and iodine.

The term “amine” as used herein refers to a —NH₂ group wherein one ormore hydrogen can be optionally substituted by a R group. R can beindependently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,or (heteroalicyclyl)alkyl.

The term “aldehyde” as used herein refers to a —R_(c)—C(O)H group,wherein R_(c) can be absent or independently selected from alkylene,alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene,arylene, heteroarylene, heteroalicyclylene, aralkylene, or(heteroalicyclyl)alkylene.

The term “amino” as used herein refers to a —NH₂ group.

The term “hydroxy” as used herein refers to a —OH group.

The term “cyano” group as used herein refers to a “—CN” group.

The term “azido” as used herein refers to a —N₃ group.

The term “thiol” as used herein refers to a —SH group.

The term “carboxylic acid” as used herein refers to —C(O)OH.

The term “thiocyanate” as used herein refers to —S—CN group.

The term “oxo-amine” as used herein refers to —O—NH₂ group, wherein oneor more hydrogen of the —NH₂ can be optionally substituted by a R group.R can be independently hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,or (heteroalicyclyl)alkyl.

As used herein, a “nucleotide” includes a nitrogen containingheterocyclic base, a sugar, and one or more phosphate groups. They aremonomeric units of a nucleic acid sequence. In RNA, the sugar is aribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxyl groupthat is present in ribose. The nitrogen containing heterocyclic base canbe purine or pyrimidine base. Purine bases include adenine (A) andguanine (G), and modified derivatives or analogs thereof. Pyrimidinebases include cytosine (C), thymine (T), and uracil (U), and modifiedderivatives or analogs thereof. The C-1 atom of deoxyribose is bonded toN-1 of a pyrimidine or N-9 of a purine.

As used herein, a “nucleoside” is structurally similar to a nucleotide,but is missing the phosphate moieties. An example of a nucleosideanalogue would be one in which the label is linked to the base and thereis no phosphate group attached to the sugar molecule. The term“nucleoside” is used herein in its ordinary sense as understood by thoseskilled in the art. Examples include, but are not limited to, aribonucleoside comprising a ribose moiety and a deoxyribonucleosidecomprising a deoxyribose moiety. A modified pentose moiety is a pentosemoiety in which an oxygen atom has been replaced with a carbon and/or acarbon has been replaced with a sulfur or an oxygen atom. A “nucleoside”is a monomer that can have a substituted base and/or sugar moiety.Additionally, a nucleoside can be incorporated into larger DNA and/orRNA polymers and oligomers.

The term “purine base” is used herein in its ordinary sense asunderstood by those skilled in the art, and includes its tautomers.Similarly, the term “pyrimidine base” is used herein in its ordinarysense as understood by those skilled in the art, and includes itstautomers. A non-limiting list of optionally substituted purine-basesincludes purine, adenine, guanine, hypoxanthine, xanthine, alloxanthine,7-alkylguanine (e.g. 7-methylguanine), theobromine, caffeine, uric acidand isoguanine. Examples of pyrimidine bases include, but are notlimited to, cytosine, thymine, uracil, 5,6-dihydrouracil and5-alkylcytosine (e.g., 5-methylcytosine).

As used herein, “derivative” or “analogue” means a synthetic nucleotideor nucleoside derivative having modified base moieties and/or modifiedsugar moieties. Such derivatives and analogs are discussed in, e.g.,Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al.,Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprisemodified phosphodiester linkages, including phosphorothioate,phosphorodithioate, alkyl-phosphonate, phosphoranilidate andphosphoramidate linkages. “Derivative”, “analog” and “modified” as usedherein, may be used interchangeably, and are encompassed by the terms“nucleotide” and “nucleoside” defined herein.

As used herein, the term “phosphate” is used in its ordinary sense asunderstood by those skilled in the art, and includes its protonatedforms (for example,

As used herein, the terms “monophosphate,” “diphosphate,” and“triphosphate” are used in their ordinary sense as understood by thoseskilled in the art, and include protonated forms.

The terms “protecting group” and “protecting groups” as used hereinrefer to any atom or group of atoms that is added to a molecule in orderto prevent existing groups in the molecule from undergoing unwantedchemical reactions. Sometimes, “protecting group” and “blocking group”can be used interchangeably.

As used herein, the prefixes “photo” or “photo-” mean relating to lightor electromagnetic radiation. The term can encompass all or part of theelectromagnetic spectrum including, but not limited to, one or more ofthe ranges commonly known as the radio, microwave, infrared, visible,ultraviolet, X-ray or gamma ray parts of the spectrum. The part of thespectrum can be one that is blocked by a metal region of a surface suchas those metals set forth herein. Alternatively or additionally, thepart of the spectrum can be one that passes through an interstitialregion of a surface such as a region made of glass, plastic, silica, orother material set forth herein. In particular embodiments, radiationcan be used that is capable of passing through a metal. Alternatively oradditionally, radiation can be used that is masked by glass, plastic,silica, or other material set forth herein.

As used herein, the term “phasing” refers to phenomena in SBS that iscaused by incomplete removal of the 3′ terminators and fluorophores, andfailure to complete the incorporation of a portion of DNA strands withinclusters by polymerases at a given sequencing cycle. Pre-phasing iscaused by the incorporation of nucleotides without effective 3′terminators and the incorporation event goes 1 cycle ahead. Phasing andpre-phasing cause the extracted intensities for a specific cycle toconsist of the signal of the current cycle as well as noise from thepreceding and following cycles. As the number of cycles increases, thefraction of sequences per cluster affected by phasing increases,hampering the identification of the correct base. Pre-phasing can becaused by the presence of a trace amount of unprotected or unblocked3′-OH nucleotides during sequencing by synthesis (SBS). The unprotected3′-OH nucleotides could be generated during the manufacturing processesor possibly during the storage and reagent handling processes.Accordingly, the discovery of nucleotide analogues which decrease theincidence of pre-phasing is surprising and provides a great advantage inSBS applications over existing nucleotide analogues. For example, thenucleotide analogues provided can result in faster SBS cycle time, lowerphasing and pre-phasing values, and longer sequencing read length.

3′-OH Protecting Groups —C(R)₂N₃

Some embodiments described herein relate to a modified nucleotide ornucleoside molecule having a removable 3′-hydroxy protecting group—C(R)₂N₃, wherein R is selected from the group consisting of hydrogen,—C(R¹)_(m)(R²)_(n), —C(═O)OR³, —C(═O)NR⁴R⁵, —C(R⁶)₂O(CH₂)_(p)NR⁷R⁸ and—C(R⁹)₂O-Ph-C(═O)NR¹⁰R¹¹, wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹⁰, R¹¹, m, n and p are defined above.

In some embodiments, one of R is hydrogen and the other R is—C(R¹)^(m)(R²)_(n). In some such embodiments, —C(R¹)_(m)(R²)_(n) isselected from —CHF₂, —CH₂F, —CHCl₂ or —CH₂Cl. In one embodiment,—C(R¹)_(m)(R²)_(n) is —CHF₂. In another embodiment, —C(R¹)_(m)(R²)_(n)is —CH₂F.

In some embodiments, one of R is hydrogen and the other R is —C(═O)OR³.In some such embodiment, R³ is hydrogen.

In some embodiments, one of R is hydrogen and the other R is—C(═O)NR⁴R⁵. In some such embodiments, both R⁴ and R⁵ are hydrogen. Insome other such embodiments, R⁴ is hydrogen and R⁵ is C₁₋₆ alkyl. Instill some other embodiments, both R⁴ and R⁵ are C₁₋₆ alkyl. In oneembodiment, R⁵ is n-butyl. In another embodiment, both R⁴ and R⁵ aremethyl.

In some embodiments, one of R is hydrogen and the other R is—C(R⁶)₂O(CH₂)_(p)NR⁷R⁸. In some such embodiments, both R⁶ are hydrogen.In some such embodiments, both R⁷ and R⁸ are hydrogen. In some suchembodiment, p is 0. In some other such embodiment, p is 6.

In some embodiments, one of R is hydrogen and the other R is—C(R⁹)₂O-Ph-C(═O)NR¹⁰R¹¹. In some such embodiments, both R⁹ arehydrogen. In some such embodiments, both R¹⁰ and R¹¹ are hydrogen. Insome other such embodiments, R¹⁰ is hydrogen and R¹¹ is a substitutedalkyl. In one embodiment, R¹¹ is an amino substituted alkyl.

Deprotection of the 3′-OH Protecting Groups

In some embodiments, the 3′-OH protecting group is removed in adeprotecting reaction with a phosphine. The azido group in —C(R)₂N₃ canbe converted to an amino group by contacting the modified nucleotide ornucleoside molecules with a phosphine. Alternatively, the azido group in—C(R)₂N₃ may be converted to an amino group by contacting such moleculeswith the thiols, in particular water-soluble thiols such asdithiothreitol (DTT). In one embodiment, the phosphine istris(hydroxymethyl)phosphine (THP). Unless indicated otherwise, thereference to nucleotides is also intended to be applicable tonucleosides.

Detectable Labels

Some embodiments described herein relate to the use of conventionaldetectable labels. Detection can be carried out by any suitable method,including fluorescence spectroscopy or by other optical means. Thepreferred label is a fluorophore, which, after absorption of energy,emits radiation at a defined wavelength. Many suitable fluorescentlabels are known. For example, Welch et al. (Chem. Eur. J. 5(3):951-960,1999) discloses dansyl-functionalised fluorescent moieties that can beused in the present invention. Zhu et al. (Cytometry 28:206-211, 1997)describes the use of the fluorescent labels Cy3 and Cy5, which can alsobe used in the present invention. Labels suitable for use are alsodisclosed in Prober et al. (Science 238:336-341, 1987); Connell et al.(BioTechniques 5(4):342-384, 1987), Ansorge et al. (Nucl. Acids Res.15(11):4593-4602, 1987) and Smith et al. (Nature 321:674, 1986). Othercommercially available fluorescent labels include, but are not limitedto, fluorescein, rhodamine (including TMR, texas red and Rox), alexa,bodipy, acridine, coumarin, pyrene, benzanthracene and the cyanins.

Multiple labels can also be used in the present application, forexample, bi-fluorophore FRET cassettes (Tet. Let. 46:8867-8871, 2000).Multi-fluor dendrimeric systems (J. Am. Chem. Soc. 123:8101-8108, 2001)can also be used. Although fluorescent labels are preferred, other formsof detectable labels will be apparent as useful to those of ordinaryskill in the art. For example, microparticles, including quantum dots(Empodocles et al., Nature 399:126-130, 1999), gold nanoparticles(Reichert et al., Anal. Chem. 72:6025-6029, 2000) and microbeads(Lacoste et al., Proc. Natl. Acad. Sci USA 97(17):9461-9466, 2000) canall be used.

Multi-component labels can also be used in the present application. Amulti-component label is one which is dependent on the interaction witha further compound for detection. The most common multi-component labelused in biology is the biotin-streptavidin system. Biotin is used as thelabel attached to the nucleotide base. Streptavidin is then addedseparately to enable detection to occur. Other multi-component systemsare available. For example, dinitrophenol has a commercially availablefluorescent antibody that can be used for detection.

Unless indicated otherwise, the reference to nucleotides is alsointended to be applicable to nucleosides. The present application willalso be further described with reference to DNA, although thedescription will also be applicable to RNA, PNA, and other nucleicacids, unless otherwise indicated.

Linkers

In some embodiments described herein, the purine or pyrimidine base ofthe modified nucleotide or nucleoside molecules can be linked to adetectable label as described above. In some such embodiments, thelinkers used are cleavable. The use of a cleavable linker ensures thatthe label can, if required, be removed after detection, avoiding anyinterfering signal with any labeled nucleotide or nucleosideincorporated subsequently.

In some other embodiments, the linkers used are non-cleavable. Since ineach instance where a labeled nucleotide of the invention isincorporated, no nucleotides need to be subsequently incorporated andthus the label need not be removed from the nucleotide.

Those skilled in the art will be aware of the utility ofdideoxynucleoside triphosphates in so-called Sanger sequencing methods,and related protocols (Sanger-type), which rely upon randomizedchain-termination at a particular type of nucleotide. An example of aSanger-type sequencing protocol is the BASS method described by Metzker.

Sanger and Sanger-type methods generally operate by the conducting of anexperiment in which eight types of nucleotides are provided, four ofwhich contain a 3′-OH group; and four of which omit the OH group andwhich are labeled differently from each other. The nucleotides usedwhich omit the 3′-OH group-dideoxy nucleotides (ddNTPs). As known by oneskilled in the art, since the ddNTPs are labeled differently, bydetermining the positions of the terminal nucleotides incorporated, andcombining this information, the sequence of the target oligonucleotidemay be determined.

The nucleotides of the present application, it will be recognized, maybe of utility in Sanger methods and related protocols since the sameeffect achieved by using ddNTPs may be achieved by using the 3′-OHprotecting groups described herein: both prevent incorporation ofsubsequent nucleotides.

Moreover, it will be appreciated that monitoring of the incorporation of3′-OH protected nucleotides may be determined by use of radioactive ³²Pin the phosphate groups attached. These may be present in either theddNTPs themselves or in the primers used for extension.

Cleavable linkers are known in the art, and conventional chemistry canbe applied to attach a linker to a nucleotide base and a label. Thelinker can be cleaved by any suitable method, including exposure toacids, bases, nucleophiles, electrophiles, radicals, metals, reducing oroxidizing agents, light, temperature, enzymes etc. The linker asdiscussed herein may also be cleaved with the same catalyst used tocleave the 3′-O-protecting group bond. Suitable linkers can be adaptedfrom standard chemical protecting groups, as disclosed in Greene & Wuts,Protective Groups in Organic Synthesis, John Wiley & Sons. Furthersuitable cleavable linkers used in solid-phase synthesis are disclosedin Guillier et al. (Chem. Rev. 100:2092-2157, 2000).

The use of the term “cleavable linker” is not meant to imply that thewhole linker is required to be removed from, e.g., the nucleotide base.Where the detectable label is attached to the base, the nucleosidecleavage site can be located at a position on the linker that ensuresthat part of the linker remains attached to the nucleotide base aftercleavage.

Where the detectable label is attached to the base, the linker can beattached at any position on the nucleotide base provided thatWatson-Crick base pairing can still be carried out. In the context ofpurine bases, it is preferred if the linker is attached via the7-position of the purine or the preferred deazapurine analogue, via an8-modified purine, via an N-6 modified adenosine or an N-2 modifiedguanine. For pyrimidines, attachment is preferably via the 5-position oncytosine, thymidine or uracil and the N-4 position on cytosine.

A. Electrophilically Cleaved Linkers

Electrophilically cleaved linkers are typically cleaved by protons andinclude cleavages sensitive to acids. Suitable linkers include themodified benzylic systems such as trityl, p-alkoxybenzyl esters andp-alkoxybenzyl amides. Other suitable linkers includetert-butyloxycarbonyl (Boc) groups and the acetal system.

The use of thiophilic metals, such as nickel, silver or mercury, in thecleavage of thioacetal or other sulfur-containing protecting groups canalso be considered for the preparation of suitable linker molecules.

B. Nucleophilic Ally Cleaved Linkers

Nucleophilic cleavage is also a well recognised method in thepreparation of linker molecules. Groups such as esters that are labilein water (i.e., can be cleaved simply at basic pH) and groups that arelabile to non-aqueous nucleophiles, can be used. Fluoride ions can beused to cleave silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or t-butyldimethyl silane (TBDMS).

C. Photocleavable Linkers

Photocleavable linkers have been used widely in carbohydrate chemistry.It is preferable that the light required to activate cleavage does notaffect the other components of the modified nucleotides. For example, ifa fluorophore is used as the label, it is preferable if this absorbslight of a different wavelength to that required to cleave the linkermolecule. Suitable linkers include those based on O-nitrobenzylcompounds and nitroveratryl compounds. Linkers based on benzoinchemistry can also be used (Lee et al., J. Org. Chem. 64:3454-3460,1999).

D. Cleavage Under Reductive Conditions

There are many linkers known that are susceptible to reductive cleavage.Catalytic hydrogenation using palladium-based catalysts has been used tocleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction isalso known in the art.

E. Cleavage Under Oxidative Conditions

Oxidation-based approaches are well known in the art. These includeoxidation of p-alkoxybenzyl groups and the oxidation of sulfur andselenium linkers. The use of aqueous iodine to cleave disulfides andother sulfur or selenium-based linkers is also within the scope of theinvention.

F. Safety-Catch Linkers

Safety-catch linkers are those that cleave in two steps. In a preferredsystem the first step is the generation of a reactive nucleophiliccenter followed by a second step involving an intra-molecularcyclization that results in cleavage. For example, levulinic esterlinkages can be treated with hydrazine or photochemistry to release anactive amine, which can then be cyclised to cleave an ester elsewhere inthe molecule (Burgess et al., J. Org. Chem. 62:5165-5168, 1997).

G. Cleavage by Elimination Mechanisms

Elimination reactions can also be used. For example, the base-catalysedelimination of groups such as Fmoc and cyanoethyl, andpalladium-catalysed reductive elimination of allylic systems, can beused.

In some embodiments, the linker can comprise a spacer unit. The lengthof the linker is unimportant provided that the label is held asufficient distance from the nucleotide so as not to interfere with anyinteraction between the nucleotide and an enzyme.

In some embodiments, the linker may consist of the similar functionalityas the 3′-OH protecting group. This will make the deprotection anddeprotecting process more efficient, as only a single treatment will berequired to remove both the label and the protecting group. Particularlypreferred linkers are phosphine-cleavable azide containing linkers.

Sequencing Methods

The modified nucleosides or nucleotides described herein can be used inconjunction with a variety of sequencing techniques. In someembodiments, the process to determine the nucleotide sequence of atarget nucleic acid can be an automated process.

The nucleotide analogues presented herein can be used in a sequencingprocedure, such as a sequencing-by-synthesis (SBS) technique. Briefly,SBS can be initiated by contacting the target nucleic acids with one ormore labeled nucleotides, DNA polymerase, etc. Those features where aprimer is extended using the target nucleic acid as template willincorporate a labeled nucleotide that can be detected. Optionally, thelabeled nucleotides can further include a reversible terminationproperty that terminates further primer extension once a nucleotide hasbeen added to a primer. For example, a nucleotide analog having areversible terminator moiety can be added to a primer such thatsubsequent extension cannot occur until a deblocking agent is deliveredto remove the moiety. Thus, for embodiments that use reversibletermination, a deblocking reagent can be delivered to the flow cell(before or after detection occurs). Washes can be carried out betweenthe various delivery steps. The cycle can then be repeated n times toextend the primer by n nucleotides, thereby detecting a sequence oflength n. Exemplary SBS procedures, fluidic systems and detectionplatforms that can be readily adapted for use with an array produced bythe methods of the present disclosure are described, for example, inBentley et al., Nature 456:53-59 (2008), WO 04/018497; WO 91/06678; WO07/123744; U.S. Pat. Nos. 7,057,026; 7,329,492; 7,211,414; 7,315,019 or7,405,281, and US Pat. App. Pub. No. 2008/0108082 A1, each of which isincorporated herein by reference.

Other sequencing procedures that use cyclic reactions can be used, suchas pyrosequencing. Pyrosequencing detects the release of inorganicpyrophosphate (PPi) as particular nucleotides are incorporated into anascent nucleic acid strand (Ronaghi, et al., Analytical Biochemistry242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi etal. Science 281(5375), 363 (1998); U.S. Pat. Nos. 6,210,891; 6,258,568and 6,274,320, each of which is incorporated herein by reference). Inpyrosequencing, released PPi can be detected by being converted toadenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATPcan be detected via luciferase-produced photons. Thus, the sequencingreaction can be monitored via a luminescence detection system.Excitation radiation sources used for fluorescence based detectionsystems are not necessary for pyrosequencing procedures. Useful fluidicsystems, detectors and procedures that can be used for application ofpyrosequencing to arrays of the present disclosure are described, forexample, in WIPO Pat. App. Ser. No. PCT/US11/57111, US Pat. App. Pub.No. 2005/0191698 A1, U.S. Pat. Nos. 7,595,883, and 7,244,559, each ofwhich is incorporated herein by reference.

Sequencing-by-ligation reactions are also useful including, for example,those described in Shendure et al. Science 309:1728-1732 (2005); U.S.Pat. Nos. 5,599,675; and 5,750,341, each of which is incorporated hereinby reference. Some embodiments can include sequencing-by-hybridizationprocedures as described, for example, in Bains et al., Journal ofTheoretical Biology 135(3), 303-7 (1988); Drmanac et al., NatureBiotechnology 16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773(1995); and WO 1989/10977, each of which is incorporated herein byreference. In both sequencing-by-ligation andsequencing-by-hybridization procedures, nucleic acids that are presentin gel-containing wells (or other concave features) are subjected torepeated cycles of oligonucleotide delivery and detection. Fluidicsystems for SBS methods as set forth herein, or in references citedherein, can be readily adapted for delivery of reagents forsequencing-by-ligation or sequencing-by-hybridization procedures.Typically, the oligonucleotides are fluorescently labeled and can bedetected using fluorescence detectors similar to those described withregard to SBS procedures herein or in references cited herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. For example, nucleotide incorporations canbe detected through fluorescence resonance energy transfer (FRET)interactions between a fluorophore-bearing polymerase andγ-phosphate-labeled nucleotides, or with zeromode waveguides. Techniquesand reagents for FRET-based sequencing are described, for example, inLevene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett.33, 1026-1028 (2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105,1176-1181 (2008), the disclosures of which are incorporated herein byreference.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in US Pat. App. Pub. Nos.2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1,each of which is incorporated herein by reference.

EXAMPLES

Additional embodiments are disclosed in further detail in the followingexamples, which are not in any way intended to limit the scope of theclaims. The synthesis of various modified nucleotide with protected3′-hydroxy group are demonstrated in Examples 1-3.

Example 1 Synthesis of Nucleotides with 3′-OH Protecting Group

Scheme 1 illustrates a synthetic route for the preparation of themodified nucleotides with monofluoromethyl substituted azidomethyl as3′-OH protecting groups. Compounds 1a-1f employ a modified thymine(T-PA) as the base. Other non-limiting examples of the bases that can beused include Cbz-PA, ADMF-PA, and GPac-PA, the structures of which areshown above in Scheme 1.

Experimental Procedures

To a solution of the starting nucleoside 1a (1.54 g, 2.5 mmol) inanhydrous CH₃CN (25 ml) was added 2,6-lutidine (0.87 mL, 7.5 mmol),(2-fluoroethyl)(4-methoxyphenyl)sulfane (MPSF) (3.26 g, 17.5 mmol) andthen Bz₂O₂ (50% pure, 8.47 g, 17.5 mmol) at 4° C. The reaction mixturewas allowed to warm up slowly to room temperature. The mixture wasstirred for other 6 hours. TLC monitored (EtOAc:DCM=2:8 v/v) to seecomplete consumption of the starting nucleoside. The reaction was thenconcentrated under reduced pressure to oily residue. To this mixture,petroleum ether (500 ml) was added and stirred vigorously for 10 min.The petroleum ether layer was decanted and the residue was repeated totreat with petroleum ether (×2). The oily residue was partitionedbetween DCM/NaHCO₃ (1:1) (300 mL). The organic layer was separated andthe aqueous was further extracted into DCM (2×150 mL). Combined organiclayers were dried over MgSO₄, filtered and the volatiles evaporatedunder reduced pressure. Crude product 1c was purified by Biotag silicagel column (50 g) using a gradient of petroleum ether to petroleumether:EtOAc 1:1 (v/v) to afford 1.63 g nucleoside 1b as a pale yellowfoam (diastereomers, 82% yield). ¹H NMR (d₆ DMSO, 400 MHz): δ, 0.95 (s,9H, tBu), 2.16-2.28 (m, 2H, H-2′), 3.67 (s, OMe), 3.65-3.85 (m, 2H,HH-5′), 3.77 (dd, J=11.1, 4.5 Hz, 1H, HH-5′), 3.95-3.98 (m, 1H, H-4′),4.04 (m, 2H, CH₂F), 4.63-4.64 (m, 1H, H-3′), 5.01-5.32 (s, 1H, CH), 6.00(m, 1H, H-1′), 6.72-6.87 (m, 3H, Ar), 7.35-7.44 (m, 7H, Ar), 7.55-7.60(m, 4H, Ar), 7.88 (s, 1H, H-6), 9.95 (brt, 1H, NH), 11.70 (s, 1H, NH).

To a solution of the starting nucleoside 1b (1.14 g, 1.4 mmol) inanhydrous CH₂C₁₂ (14 mL) with molecular sieve (4 Å) under N₂ was addedcyclohexene (1.44 mL, 14 mmol). The mixture was cooled with a dryice/acetone bath to −78° C. The solution of sulfuryl chloride (580 μL,7.2 mmol) in DCM (14 ml) was slowly added over 90 minutes under N₂.After 20 mins at that temperature TLC (EtOAc:petroleum ether=1:1 v/v)indicated the full consumption of the starting nucleoside. Volatileswere evaporated under reduced pressure (and room temperature of 25° C.)and the oily residue was quickly subjected to high vacuum for a further10 minutes until it foamed. The crude product was purged with N₂ andthen dissolved in anhydrous DMF (5 mL) and NaN₃ (470 mg, 7 mmol) addedat once. The resulting suspension was stirred at room temperature for 2hours or until TLC indicated the completion of the reaction andformation of 1c as two isomer (a and b) The reaction mixture waspartitioned between EtOAc:NaHCO₃ (1:1) (200 mL). The organic layer wasseparated and the aqueous was further extracted into EtOAc (2×100 mL).Combined organic extracts were dried over MgSO₄, filtered and thevolatiles evaporated under reduced pressure. The two diastereoisomers of1c (A and B) were separated by Biotag silica gel column (25 g) using agradient of petroleum ether to petroleum ether:EtOAc 1:1 (v/v) as paleyellow foam.

Isomer A (370 mg, yield: 38%). ¹H NMR (d₆ DMSO, 400 MHz): δ 1.02 (s, 9H,tBu), 2.35-2.43 (m, 2H, H-2′), 3.76-3.80 (m, 1H, H-5′), 3.88-3.92 (m,1H, H-5′), 4.10-4.12 (m, 1H, H-4′), 4.14 (d, J=4.1 Hz 2H, NHCH₂),4.46-4.60 (m, 3H, H-3′, CH₂F), 5.05-5.09 (m, 1H, CHN₃), 6.11 (t, J=6.1Hz, 1H, H-1′), 7.47-7.51 (m, 6H, Ar), 7.64-7.68 (m, 4H, Ar), 7.97 (s,1H, H-6), 10.03 (bt, 1H, J=10.0 Hz, NH), 11.76 (s, 1H, NH). ¹⁹F NMR:−74.3 (CF₃), −230.2 (CH₂F).

Isomer B (253 mg, yield: 26%). ¹H NMR (d₆ DMSO, 400 MHz): δ 1.01 (s, 9H,tBu), 2.38-2.42 (m, 2H, H-2′), 3.74-3.78 (m, 1H, H-5′), 3.86-3.90 (m,1H, H-5′), 4.00-4.05 (m, 1H, H-4′), 4.12 (d, J=4.1 Hz 2H, NHCH₂),4.45-4.60 (m, 3H, H-3′, CH₂F), 5.00-5.14 (m, 1H, CHN₃), 6.09 (t, J=6.1Hz, 1H, H-1′), 7.41-7.50 (m, 6H, Ar), 7.63-7.66 (m, 4H, Ar), 7.95 (s,1H, H-6), 10.01 (bs, 1H, NH), 11.74 (s, 1H, NH). ¹⁹F NMR: −74.5 (CF₃),−230.4 (CH₂F).

The starting material 1c (isomer A) (500 mg, 0.71 mmol) was dissolved inTHF (3 mL) and cooled to 4° C. in ice-bath. Then TBAF (1.0 M in THF, 5wt. % water, 1.07 mL, 1.07 mmol) was added slowly over a period of 5mins. The reaction mixture was slowly warmed up to room temperature.Reaction progress was monitored by TLC (petroleum ether:EtOAc 3:7(v/v)). The reaction was stopped after 1 hour when no more startingmaterial was visible by TLC. The reaction solution was dissolved inEtOAc (50 mL) and added to NaHCO₃ (60 mL). The two layers were separatedand the aqueous layer was extracted with additional DCM (50 mL×2). Theorganic extractions were combined, dried (MgSO₄), filtered, andevaporated to give a yellow oil. Crude product 1d (isomer A) waspurified by Biotag silica gel column (10 g) using a gradient ofpetroleum ether:EtOAc 8:2 (v/v) to EtOAc as a white solid (183 mg,yield: 56%).

Isomer A: ¹H NMR (400 MHz, d₆-DMSO): δ 2.24-2.35 (m, 2H, H-2′),3.56-3.66 (m, 2H, H-5′), 3.96-4.00 (m, 1H, H-4′), 4.23 (s, 2H, CH₂NH),4.33-4.37 (m, 1H, H-3′), 4.43-4.51 (m, CH₂F), 5.12 (br.s, 1H, CHN₃),5.23 (br.s, 1H, 5′-OH), 6.07 (t, J=6.7 Hz, 1H, H-1′), 8.26 (s, 1H, H-6),10.11 (br s, 1H, NH), 11.72 (br s, 1H, NH). ¹⁹F NMR: −74.3 (CF₃), −230.5(CH₂F)

The same reaction was performed for 1c (isomer B) at 360 mg scale andafforded the corresponding product 1d (Isomer B, 150 mg, 63%). ¹H NMR(400 MHz, d₆-DMSO): δ 2.24-2.37 (m, 2H, H-2′), 3.57-3.70 (m, 2H, H-5′),3.97-4.01 (m, 1H, H-4′), 4.23 (br.s, 2H, CH₂NH), 4.33-4.37 (m, 1H,H-3′), 4.44-4.53 (m, CH₂F), 5.11-5.21 (br.s, 1H, CHN₃), 5.23 (br.s, 1H,5′-OH), 6.07 (t, J=6.6 Hz, 1H, H-1′), 8.23 (s, 1H, H-6), 10.09 (br s,1H, NH), 11.70 (br s, 1H, NH). ¹⁹F NMR: −74.1 (CF₃), −230.1 (CH₂F).

The preparation of the corresponding triphosphates 1e and the furtherattachment of dye to the nucleobase to afford the fully functionalnucleoside triphosphate (ffN) if have been reported in WO 2004/018497and are generally known by one skilled in the art.

Example 2 Synthesis of Nucleotides with 3′-OH Protecting Group

Scheme 2 illustrates a synthetic route for the preparation of themodified nucleotides with C-amido substituted azidomethyl as 3′-OHprotecting groups. Compounds 2a-2i employ a modified thymine (T-PA) asthe base. Other non-limiting examples of the bases that can be usedinclude Cbz-PA, ADMF-PA, and GPac-PA, the structures of which are shownabove in Scheme 1. In the experimental procedure, compound 2f with aN,N-dimethyl-C(═O)— substituted azidomethyl protecting group (R═NMe₂)and the subsequent reactions were reported. Compounds with other C-amidogroups were also prepared, such as N-ethyl-C(═O)— (R=NHEt).

Experimental Procedures

To a solution of the starting nucleoside 2a (4.27 g, 6.9 mmol) inanhydrous CH₃CN (50 ml) was added 2,6-lutidine (2.4 mL, 20.7 mmol),S(CH₂CH₂OAc)₂ (12.2 g, 69 mmol) and then Bz₂O₂ (50% pure, 33.4 g, 69mmol) at 4° C. The reaction mixture was allowed to warm up slowly toroom temperature. The mixture was stirred for other 12 hours. TLCmonitored (EtOAc:DCM=4:6 v/v) to see complete consumption of thestarting nucleoside. The reaction was then concentrated under reducedpressure to an oily residue. To this mixture, petroleum ether (800 ml)was added and stirred vigorously for 10 min. The petroleum ether layerwas decanted and the residue was repeatedly treated with petroleum ether(×2). The oily residue was then partitioned between DCM/NaHCO₃ (1:1)(1000 mL). The organic layer was separated and the aqueous layer wasfurther extracted into DCM (2×500 mL). Combined organic layers weredried over MgSO₄, filtered and the volatiles evaporated under reducedpressure. Crude product 2b was purified by a Biotag silica gel column(100 g) using a gradient of petroleum ether to petroleum ether:EtOAc 2:8(v/v) as a pale yellow foam (4.17 g, yield: 74%, diastereoisomers).

To a solution of the starting nucleoside 2b (4.54 g, 5.56 mmol) inanhydrous CH₂C₁₂ (56 mL) with molecular sieve (4 Å) under N₂ was addedcyclohexene (5.62 mL, 56 mmol). The mixture was cooled with an ice bathto 4° C. The solution of sulfuryl chloride (1.13 mL, 13.9 mmol) in DCM(25 ml) was slowly added over 90 minutes under N₂. After 30 min at thattemperature TLC (EtOAc:DCM=4:6 v/v) indicated 10% of the startingnucleoside 2b was left. Additional sulfuryl chloride (0.1 mL) was addedinto reaction mixture. TLC indicated complete conversion of 2b.Volatiles were evaporated under reduced pressure (and room temperatureof 25° C.) and the oily residue was quickly subjected to a high vacuumfor a further 10 minutes until it foamed. The crude product was purgedwith N₂ and then dissolved in anhydrous DMF (5 mL) and NaN₃ (1.8 g, 27.8mmol) added at once. The resulting suspension was stirred at roomtemperature for 2 hours or until TLC indicated the completion of thereaction and formation of 2c as two isomers (A and B). The reactionmixture was partitioned between EtOAc:NaHCO₃ (1:1) (1000 mL). Theorganic layer was separated and the aqueous layer was further extractedinto EtOAc (2×300 mL). Combined organic extracts were then dried overMgSO₄, filtered and the volatiles evaporated under reduced pressure. Thetwo diastereoisomers 2c (isomer A and B) were separated by a Biotagsilica gel column (100 g) using a gradient of petroleum ether topetroleum ether:EtOAc 1:1 (v/v) as pale yellow foam. Isomer A: 1.68 g,yield: 40.7%. Isomer B: 1.79 g, yield: 43.2%.

To a solution of the starting nucleoside 2c (isomer A) (1.63 g, 2.2mmol) in MeOH/THF (1:1) (20 mL) was slowly added NaOH (1M in water) (2.2mL, 2.2 mmol) and stirred in 4° C. The reaction progress was monitoredby TLC (EtOAc:DCM=4:6 v/v). The reaction was stopped after 1 hour whenno more starting material was visible by TLC. The reaction mixture waspartitioned between DCM:NaHCO₃ (1:1) (150 mL). The organic layer wasseparated and the aqueous layer was further extracted into DCM (2×70mL). Combined organic extracts were dried over MgSO₄, filtered and thevolatiles evaporated under reduced pressure. The crude product 2d waspurified by a Biotag silica gel column (10 g) using a gradient ofpetroleum ether:EtOAc (8:2) (v/v) to EtOAc as a pale yellow foam (1.1 g,yield: 71%).

The same reaction was repeated for 2c (isomer B, 1.57 g) and affordedthe corresponding product 2d (isomer B, 1.01 g, 69% yield).

To a solution of the starting nucleoside 2d (isomer A) (700 mg, 1 mmol)in CH₃CN (10 mL) was treated with TEMPO (63 mg, 0.4 mmol) and BAIB (644mg, 2 mmol) at room temperature. The reaction progress was monitored byTLC (EtOAc:DCM=7:3 v/v). The reaction was stopped after 2 hour when nomore starting material was visible by TLC. The reaction mixture waspartitioned between DCM:Na₂S₂O₃ (1:1) (100 mL). The organic layer wasseparated and the aqueous layer was further extracted into DCM (2×70mL). Combined organic extracts were then washed with NaCl (sat.). Theorganic layer was evaporated under reduced pressure without drying overMgSO₄ in order to prevent the product from precipitating out. The crudeproduct 2e was purified by a Biotag silica gel column (10 g) using agradient of petroleum ether:EtOAc (1:1) (v/v) to EtOAc to MeOH:EtOAc(1:9) as a pale yellow foam (isomer A, 482 mg, 68% yield).

The same reaction was performed for 2d (isomer B, 700 mg) and affordedthe corresponding product 2e (isomer B, 488 mg, 69% yield).

To a solution of the starting nucleoside 2e (isomer A) (233 mg, 0.33mmol) in CH₃CN (10 mL) was added Hunig's base (173 μL, 1 mmol) and BOP(165 mg, 0.39 mmol) at room temperature. After stirring for 5 min, thesolution was treated with Me2NH (2 M in THF) (0.41 ml, 0.82 mmol). Thereaction progress was monitored by TLC (MeOH:DCM=1:9 v/v). The reactionwas stopped after 2 hours when no more starting material was visible byTLC. The reaction mixture was partitioned between DCM:NaHCO₃ (1:1) (50mL). The organic layer was separated and the aqueous layer was furtherextracted into DCM (2×30 mL). Combined organic extracts were dried overMgSO₄, filtered and the volatiles evaporated under reduced pressure. Thecrude product 2f (R═NMe₂) was purified by a Biotag silica gel column (10g) using a gradient of DCM:EtOAc (8:2) (v/v) to EtOAc as a pale yellowfoam (isomer A, 220 mg, 90% yield).

The same reaction was performed for 2e (isomer B, 249 mg) and affordedthe corresponding product 2f (isomer B, 240 mg, 92% yield).

The starting material 2f (mixture of isomer A and B) (455 mg, 0.61 mmol)was dissolved in THF (2 mL) and cooled to 4° C. with ice-bath. Then,TBAF (1.0 M in THF, 5 wt. % water, 1.0 mL, 1.0 mmol) was added slowlyover a period of 5 min. The reaction mixture was slowly warmed up toroom temperature. The reaction progress was monitored by TLC (EtOAc).The reaction was stopped after 1 hour when no more starting material wasvisible by TLC. The reaction solution was dissolved in DCM (30 mL) andadded to NaHCO₃ (30 mL). The two layers were separated and the aqueouslayer was extracted with additional DCM (30 mL×2). The organicextractions were combined, dried (MgSO₄), filtered, and evaporated togive a yellow oil. Crude product 2 g was purified by a Biotag silica gelcolumn (10 g) using a gradient of DCM:EtOAc 8:2 (v/v) to EtOAc toMeOH:EtOAc (2:8) as a white solid (52% yield, 160 mg).

The preparation of the corresponding triphosphates 2h and the furtherattachment of dye to the nucleobase to afford the fully functionalnucleoside triphosphate (ffN) 2i have been reported in WO 2004/018497and are generally known by one skilled in the art.

Example 3 Synthesis of Nucleotides with 3′-OH Protecting Group

Scheme 3 illustrates a synthetic route for the preparation of modifiednucleotides with difluoromethyl substituted azidomethyl 3′-OH protectinggroups. Compounds 3a-3i employ a modified thymine (T-PA) as the base.Other non-limiting examples of the bases that can be used includeCbz-PA, ADMF-PA, and GPac-PA, the structures of which are shown above inScheme 1. The procedure for the synthesis of 3b, 3c and 3d weredescribed in Example 2.

Experimental Procedures

To a solution of the starting nucleoside 3d (isomer A) (490 mg, 0.7mmol) and DBU (209 μL, 1.4 mmol) in anhydrous DCM (5 mL) was addedslowly a solution of N-tert-butyl benzene sulfinimidoyl chloride (181mg, 0.84 mmol) in anhydrous DCM (2 ml) at −78° C. The reaction mixturewas stirred for 2 h at −78° C. The reaction progress was monitored byTLC (EtOAc:DCM 4:6 v/v). The reaction was stopped after 2 hours whenthere was still 10% starting material left by TLC, to preventover-reacting. The reaction mixture was partitioned between DCM:NaHCO₃(1:1) (50 mL). The aqueous layer was further extracted into DCM (2×30mL). The organic extractions were combined, dried (MgSO₄), filtered, andevaporated to give a yellow oil. The crude product 3e was purified by aBiotag silica gel column (10 g) using a gradient of petroleumether:EtOAc (8:2) (v/v) to petroleum ether:EtOAc (2:8) (v/v) as a paleyellow foam (isomer A, 250 mg, 51% yield).

The same reaction was performed for 3d (isomer B, 480 mg) and affordedthe corresponding product 3e (isomer B, 240 mg, 50% yield).

To a solution of the starting nucleoside 3e (isomer A) (342 mg, 0.49mmol), EtOH (15 μL, 0.25 mmol) in DCM (2.5 mL) was added slowly to thesolution of DAST (181 mg, 0.84 mmol) in DCM (2.5 mL) at 4° C. (icebath). The reaction mixture was stirred for 1 h at 4° C. The reactionprogress was monitored by TLC (EtOAc:petroleum ether=3:7 v/v). Thereaction was stopped after 1 hour. The reaction mixture was partitionedbetween DCM:NaHCO₃ (1:1) (50 mL). The aqueous layer was furtherextracted into DCM (2×30 mL). The organic extractions were combined,dried (MgSO₄), filtered, and evaporated to give a yellow oil. The crudeproduct 3f was purified by a Biotag silica gel column (10 g) using agradient of petroleum ether:EtOAc (9:1) (v/v) to petroleum ether:EtOAc(2:8) (v/v) as a pale yellow foam (isomer A, 100 mg, 28%).

The same reaction was performed for 3e (isomer B, 480 mg) and affordedthe corresponding product 3f (isomer B, 240 mg, 50% yield).

The starting material 3f (isomer A) (124 mg, 0.17 mmol) was dissolved inTHF (2 mL) and cooled to 4° C. with an ice bath. Then, TBAF (1.0 M inTHF, 5 wt. % water, 255 μL, 10.255 mmol) was added slowly over a periodof 5 min. The reaction mixture was slowly warmed up to room temperature.The reaction progress was monitored by TLC (EtOAc). The reaction wasstopped after 1 hour when no more starting material was visible by TLC.The reaction solution was dissolved in DCM (30 mL) and added to NaHCO₃(30 mL). The two layers were separated and the aqueous layer wasextracted with additional DCM (30 mL×2). The organic extractions werecombined, dried (MgSO₄), filtered, and evaporated to give a yellow oil.Crude product 3 g was purified by a Biotag silica gel column (4 g) usinga gradient of DCM:EtOAc 8:2 (v/v) to EtOAc to MeOH:EtOAc (2:8) as a paleyellow foam (isomer A, 54% yield, 44 mg).

Isomer A: ¹H NMR (400 MHz, d₆-DMSO): δ 2.24-2.35 (m, 2H, H-2′),3.56-3.66 (m, 2H, H-5′), 3.96-4.00 (m, 1H, H-4′), 4.23 (s, 2H, CH₂NH),4.33-4.37 (m, 1H, H-3′), 4.85 (s, 2H, OCH₂N₃), 5.23 (t, J=5.1 Hz, 1H,5′-OH), 6.07 (t, J=6.7 Hz, 1H, H-1′), 8.19 (s, 1H, H-6), 10.09 (br s,1H, NH), 11.70 (br s, 1H, NH). ¹⁹F NMR: −74.4 (CF₃), −131.6 (CH₂F).

The same reaction was performed for 3f (isomer B, 133 mg) and affordedthe corresponding product 3 g (isomer B, 48 mg, 54% yield). ¹H NMR (400MHz, d₆-DMSO): δ 2.27-2.44 (m, 2H, H-2′), 3.58-3.67 (m, 2H, H-5′),4.00-4.02 (m, 1H, H-4′), 4.24 (d, J=4.1 Hz, 2H, CH₂NH), 4.57-4.58 (m,1H, H-3′), 5.24-5.29 (m, 2H, 5′-OH, OCHN₃), 6.07-6.34 (m, 2H, H-1′,CHF₂), 8.19 (s, 1H, H-6), 10.09 (br s, 1H, NH), 11.70 (br s, 1H, NH).¹⁹F NMR: −74.2 (CF₃), −131.4 (CH₂F).

The preparation of the corresponding triphosphates 3h and the furtherattachment of dye to the nucleobase to afford the fully functionalnucleotide (ffN) 3i have been reported in WO 2004/018497 and aregenerally known by one skilled in the art.

Example 4 Thermal Stability Testing of the 3′-OH Protecting Groups

A variety of 3′-OH protecting groups were investigated in regard totheir thermal stability (FIG. 1A). The thermal stability was evaluatedby heating 0.1 mM of each 3′-OH protected nucleotide in a pH=9 buffer(tis-HCl 50 mM, NaCl 50 mM, tween 0.05%, Mg₂SO₄ 6 mM) at 60° C. Varioustimes points were taken and HPLC was used to analyze the formation ofun-blocked materials. The stabilities of —CH₂F and —C(O)NHBu were foundto be about 2-fold greater than the standard azidomethyl (—CH₂N₃)protecting group. The stability of —CF₂H group was found to be about10-fold greater than the standard (FIG. 1B).

Example 5 Deprotection of the 3′-OH Protecting Groups

The deprotecting reaction rates of several 3′-OH protecting groups werealso studied. The deprotection rate of the standard azidomethylprotecting group was compared with the —CH₂F substituted azidomethyl and—C(O)NHBu substituted azidomethyl. It was observed that both of the morethermally stable 3′-OH blocking groups were removed faster than thestandard azidomethyl protecting group using phosphines (1 mM THP) as thedeprotecting agent. See FIG. 2A. For example, the half-life of —CH₂F and—C(O)NHBu was 8.9 minutes and 2.9 minutes respectively, compared to the20.4 minutes half-life of azidomethyl (FIG. 2B).

Example 6 Sequencing Test

Modified nucleotides with —CH₂F (mono-F) substituted azidomethyl 3′-OHprotecting group were prepared and their sequencing performance wasevaluated on Miseq platforms. It was envisaged that increased thermalstability of 3′-OH protecting groups would lead to a higher quality ofnucleotides for sequencing chemistry with less contaminated 3′-unblockednucleotides. The presence of 3′-unblocked nucleotides in theSBS-sequencing kits would therefore result in pre-phasing events, whichwere numerated as pre-phasing values.

Short 12-cycle sequencing experiments were first used to generatephasing and pre-phasing values. Mono-F substituted azidomethyl protectedffNs were used according to the following concentration: ffA-dye 1 (2uM); ffT-dye 2 (10 uM), ffC-dye 3 (2 uM) and ffG-dye 4 (5 uM). Mono-Fsubstituted azidomethyl group comprises both isomer A and B. Twodyes—dye 2 as in standard Miseq kits and dye 5 were used to label ffT.Table 1 shows various nucleotide combinations with A and B isomers ofmono-F substituted azidomethyl that were evaluated in regard to phasingand pre-phasing impacts. In all cases, the pre-phasing values weresubstantially lower than the control that standard V2 Miseq kitsnucleotides used (FIG. 3).

TABLE 1 Phasing Pre-phasing Sample 3′-OH Protecting Group (%) (%) 1 StdMiseq V2 IMX control 0.119 0.177 2 Mono-F-A-isomer 0.11 0.085 3 Mono-F-Aisomer (ffT-Dye 5) 0.076 0.032 4 Mono-F-A isomer (A, C and G) + 0.0950.083 Mono-F-B-ffT-Dye 5 5 Mono-F-A (G, ffT-Dye 5) and Mono- 0.104 0.05F-B (A, C) 6 Mono-F-A (G, T) and Mono-F-B 0.098 0.095 (A, C) 7 Std MiseqV2 IMX control 0.145 0.167Sequencing Quality Testing

2×400 bp sequencing was carried out on Miseq to evaluate the potentialof these nucleotides for sequencing quality improvement. The sequencingrun was performed according to manufacturer's instructions (IIluminaInc., San Diego, Calif.). The standard incorporation buffer was replacedwith an incorporation buffer containing all mono-F blocked FFNs, eachwith a separate dye label: ffA-dye 1 (2 uM), ffT-dye 2 (1 uM), ffC-dye 3(2 uM) and ffG-dye 4 (5 uM). The DNA library used was made following thestandard TruSeq HT protocol from B. cereus genomic DNA.

In both sequencing experiments (with mono-F block A and B isomer), verylow pre-phasing values were observed. Coupled with low phasing values,application of these new nucleotides has generated superior 2×400 bpsequencing data with >80% of bases above Q30 in both cases (see FIG. 4Afor the Q score of isomer A and FIG. 4B for the Q score chart of isomerB). These results demonstrate a great improvement compared with Miseq v2kits (2×250 bp, 80% bases>Q30 in a typical R&D sequencing experiments,or 70% bases >Q30 as the stated specs). As shown below, Table 2summarizes the sequencing data when using all mono-F ffNs-A-isomer inIMX. Table 3 summarizes the sequencing data using all mono-FffNs-B-isomer in IMX.

TABLE 2 % Yield Density Clusters Phas/Prephas Reads Reads Mismatch % >=Total Lane Tiles (K/mm2) PF (%) (%) (M) PF (M) Rate (PF) Q30 (G) R1 28690 +/− 14 93.1 +/− 0.7 0.075/0.051 13.35 12.43 0.58 ± 0.11 89.7 5 R2 28690 +/− 14 93.1 +/− 0.7 0.092/0.078 13.35 12.43 1.31 ± 0.25 81.9 5 Total85.8 9.9

TABLE 3 % Yield Density Clusters Phas/Prephas Reads Reads Mismatch % >=Total Lane Tiles (K/mm2) PF (%) (%) (M) PF (M) Rate (PF) Q30 (G) R1 28816 +/− 9 92.7 +/− 0.6 0.073/0.033 15.79 14.64 0.44 ± 0.11 91.2 5.9 R228 816 +/− 9 92.7 +/− 0.6 0.078/0.059 15.79 14.64 1.03 ± 0.19 83.4 5.9Total 87.3 11.7

What is claimed is:
 1. A modified nucleotide triphosphate comprising anucleobase and a 2-deoxyribose sugar moiety having a removable3′-hydroxy protecting group forming a structure —O—CH(R)N₃ covalentlyattached to the 3′-carbon atom, wherein R is selected from the groupconsisting of —C(R¹)_(m)(R²)_(n), —C(═O)OR³, —C(═O)NR⁴R⁵,—C(R⁶)₂O(CH₂)_(p)NR⁷R⁸ and —C(R⁹)₂O-Ph-C(═O)NR¹⁰R^(ii); R¹ is hydrogen,or optionally substituted alkyl; R² is halogen; R³ is hydrogen oroptionally substituted alkyl; each of R⁴ and R⁵ is independentlyselected from the group consisting of hydrogen, optionally substitutedalkyl, optionally substituted aryl, optionally substituted heteroaryl,and optionally substituted aralkyl; each of R⁶ and R⁹ is selected fromthe group consisting of hydrogen, optionally substituted alkyl andhalogen; each of R⁷, R⁸, R¹⁰ and R¹¹ is independently selected from thegroup consisting of hydrogen, optionally substituted alkyl, optionallysubstituted aryl, optionally substituted heteroaryl, and optionallysubstituted aralkyl; m is an integer of 1 or 2; n is an integer of 1 or2; provided that m+n is equal to 3; and p is an integer of 0 to
 6. 2.The modified nucleotide triphosphate of claim 1, wherein R is —CHF₂,—CH₂F, —CHCl₂ or —CH₂Cl.
 3. The modified nucleotide triphosphate ofclaim 2, wherein R is —CH₂F or —CHF₂.
 4. The modified nucleotidetriphosphate of claim 3, wherein the nucleobase is deazapurine,cytosine, thymine, or uracil.
 5. The modified nucleotide triphosphate ofclaim 1, wherein R is —C(═O)NR⁴R⁵ and wherein R⁴ is hydrogen and R⁵ isC₁₋₆ alkyl.
 6. The modified nucleotide triphosphate of claim 5, whereinR⁵ is t-butyl.
 7. The modified nucleotide triphosphate of claim 5,wherein the nucleobase is deazapurine, cytosine, thymine, or uracil. 8.The modified nucleotide triphosphate of claim 1, wherein said nucleotideis linked to a detectable label via a cleavable linker.
 9. The modifiednucleotide triphosphate of claim 7, wherein the cleavable linker isattached to the nucleobase.
 10. The modified nucleotide triphosphate ofclaim 9, wherein the cleavable linker is attached via the 7-position ofthe deazapurine, or the 5-position of the cytosine, thymine, or uracil.11. The modified nucleotide triphosphate of claim 8, wherein thecleavable linker comprises azido group.
 12. The modified nucleotidetriphosphate of claim 8, wherein the cleavable linker and the 3′-hydroxyprotecting group are cleavable in a single step of chemical reaction.13. A method of preparing a growing polynucleotide complementary to atarget single-stranded polynucleotide in a sequencing reaction,comprising: incorporating a modified nucleotide triphosphate of claim 1into a growing complementary polynucleotide, wherein the incorporationof the modified nucleotide prevents the introduction of any subsequentnucleotide into the growing complementary polynucleotide.
 14. The methodof claim 13, wherein the incorporation of the modified nucleotide isaccomplished by a terminal transferase, a terminal polymerase or areverse transcriptase.
 15. A method of determining the sequence of atarget single-stranded polynucleotide, comprising (a) incorporating amodified nucleotide triphosphate of claim 8 into a growing complementarypolynucleotide, wherein the incorporation of the modified nucleotideprevents the introduction of any subsequent nucleotide into the growingcomplementary polynucleotide; (b) detecting the identity of the modifiednucleotide incorporated; and (c) removing the 3′-hydroxy protectinggroup and the detectable label prior to introducing the nextcomplementary nucleotide.
 16. The method of claim 15, wherein theincorporation of the modified nucleotide is accomplished by a terminalpolymerase.
 17. The method of claim 15, wherein the identity of themodified nucleotide is determined by detecting the detectable label. 18.The method of claim 15, wherein the 3′-hydroxy protecting group and thedetectable label are removed in a single step of chemical reaction. 19.The method of claim 15, wherein steps (a)-(c) are repeated multipletimes.
 20. A kit comprising one or more modified nucleotide triphosphateof claim
 1. 21. The kit of claim 20, wherein the modified nucleotide islinked to a detectable label via a cleavable linker.